Meeting Abstract
Amplified fragment length polymorphism (AFLP), a technique for genetic fingerprinting based on polymerase chain reaction (PCR), can present financial and logistical challenges, as the use of radioactive isotopes and sequencing gels can be expensive and dangerous. The goal of our research was to optimize a protocol for the genetic fingerprinting of different clone lines of pale anemones (Aiptasia pallida) that did not require the use of these materials. DNA from individual anemones was extracted and digested with both EcoRI and MseI restriction endonucleases. Adapters were ligated to these cut sites, and the resulting fragments were amplified via nested PCR using increasingly selective EcoRI- and MseI-specific primers. In the final amplification step, EcoRI primers were labeled with 5(6)-carboxyfluorescein (FAM) tags instead of radioactive isotopes. The resulting DNA fragments were electrophoresed through pre-cast 8% tris-borate-ethylenediaminetetraacetic acid (TBE) polyacrylamide mini-gels rather than sequencing gels. To facilitate band scoring, a FAM-labeled ladder was run on these gels alongside the amplified DNA samples. Gels were photographed on a variable mode imager and bands were marked manually. Fragment lengths were determined using ImageQuant TL software by comparison to the FAM-labeled ladder run on each gel. Estimates of genetic similarity between each pair of fingerprints were then calculated using the Jaccard coefficient. Comparisons of sample replicates and individuals from the same clone line resulted in consistently larger Jaccard coefficients than comparisons of independent samples. Our AFLP technique facilitates DNA fingerprinting by student researchers, allowing for a broader application of genetic fingerprinting.
